Project Summary/Abstract Chromosomally mediated resistance to ceftriaxone in Neisseria gonorrhoeae is of great concern in public health. With over 350,000 infections reported in the US, and over 78 million infections estimated world-wide, the possibility of losing the most effective antibiotic for treating gonorrhea is such a concern that the CDC has labeled multidrug resistant N. gonorrhoeae an urgent public health threat. N. gonorrhoeae has become resistant to essentially all antibiotics that have been used historically to treat gonococcal infections, including penicillin, tetracycline, ciprofloxacin, and incidences of resistance to the currently used dual therapy antibiotics, ceftriaxone and azithromycin, are rapidly increasing. Despite all of the research on antibiotics and their lethal targets, the actual mechanism by which bacteria are killed by bactericidal antibiotics is still not entirely clear. It has been proposed that bactericidal antibiotics kill Escherichia coli by a common mechanism, and our studies in N. gonorrhoeae are consistent with this hypothesis. By transforming all known resistance determinants from different resistant clinical isolates into multiple recipient strains, we have determined that whereas we can reach the same MICs of bacteriostatic antibiotics as those of the donor strains, the MICs of a broad range of bactericidal antibiotics are consistently 3-4 fold lower in the transformed strains compared to the donors. This pan-resistance suggests that bactericidal antibiotics kill bacteria by a common mechanism, and that resistant clinical isolates have altered this common mechanism to increase resistance to bactericidal antibiotics. This proposal focuses on the genetic analysis of antibiotic resistance. In Specific Aim 1, we will examine the transcriptional profile of PenR and CephR clinical isolates that display pan-resistance to bactericidal antibiotics, both in the absence and in the presence of a breakpoint concentration of a set of bactericidal and bacteriostatic antibiotics. Moreover, because in clinical settings N. gonorrhoeae never interacts with antibiotics in isolation, but within the host, these studies will also be done in the presence of human-derived endocervical epithelial cells. These studies will test the hypothesis that host cells targeted by gonococci impose stresses distinct from that experienced by gonococci in laboratory media during exposure to bactericidal antibiotics. Once this set of genes is identified, their roles in resistance to bactericidal antibiotics will be investigated. In Specific Aim 2, we will utilize transposon mutagenesis with negative selection to identify genes involved in intrinsic resistance to ?-lactam antibiotics, which will identify new targets for future drug development. The proposed studies build on our past successes in defining the mechanisms of chromosomally mediated antibiotic resistance in N. gonorrhoeae, while utilizing new technologies and approaches to identify genes that modulate resistance to antimicrobials. These studies will enable us to fully understand the complex mechanisms involved in mediating gonococcal resistance to ceftriaxone and other bactericidal antibiotics.